InteractiveFly: GeneBrief

Bicoid interacting protein 1: Biological Overview | References


Gene name - Bicoid interacting protein 1

Synonyms - Sap18

Cytological map position-89B7-89B7

Function - transcription factor

Keywords - anterior-posterior polarity

Symbol - Bin1

FlyBase ID: FBgn0024491

Genetic map position - 3R:12,011,880..12,012,615 [-]

Classification - SAP18, Sin3 associated polypeptide p18

Cellular location - nuclear



NCBI links: Precomputed BLAST | EntrezGene
BIOLOGICAL OVERVIEW

Development of the insect head is a complex process that in Drosophila requires the anterior determinant, Bicoid. Bicoid is present in an anterior-to-posterior concentration gradient, and binds DNA and stimulates transcription of head-specific genes. Many of these genes, including the gap-gene hunchback, are initially activated in a broad domain across the head primordium, but later retract so that their expression is cleared from the anterior-most segmented regions. This study shows that retraction requires Bicoid-interacting protein, also known as Sap18, which is part of the Sin3A/Rpd3 histone deacetylase complex. In sensitized-mutant backgrounds (e.g., bcdE1/+, removal of maternal sap18 results in embryos that are missing labrally derived parts of the cephalopharyngeal skeleton. These sap18 mutant embryos fail to repress hb expression, and show reduced anterior cap expression of the labral determinant cap 'n' collar. These phenotypes are enhanced by lowering the dose of rpd3, which encodes the catalytic subunit of the deacetylase complex. The results suggest a model where, in labral regions of the head, Bicoid is converted from an activator into a repressor by recruitment of a co-repressor to Bicoid-dependent promoters. Bicoid's activity, therefore, depends not only on its concentration gradient, but also on its interactions with modifier proteins within spatially restricted domains (Singh, 2005).

Although SAP18 is conserved among humans, Caenorhabditis elegans, and flies, little is known about its function. No clear ortholog exists in yeast, and there are no compelling data on its exact role in any organism. No functional motifs are revealed by its sequence. It was discovered by biochemical fractionation of human cell extracts as an 18-kDa peptide that co-purifies with the Sin3A/Rpd3 HDAC complex, and enhances Sin3-mediated repression (Zhang, 1997). Drosophila Sap18 also interacts with GAGA and E(z) proteins, which are implicated in Trithorax- and Polycomb-mediated regulation of homeotic genes, respectively (Espinas, 2000 and Wang, 2002). Mammalian SAP18 interacts with Su(Fu), which is a repressor of the Gli transcription factor in the Hedgehog signaling pathway (Cheng, 2002). In each study, SAP18 was proposed to be an adaptor protein that bridges the interaction between a DNA-binding protein and a Sin3-HDAC co-repression complex. SAP18 was also found to be part of a complex known as ASAP, which contains both an RNA-binding protein (RNPS1) and a caspase (Acinus) (Schwerk, 2003), suggesting that it plays a role in RNA processing and apoptosis (Singh, 2005 and references therein).

This paper tested whether the interaction of Bicoid with Sap18 is important for embryonic head development, and whether Sap18 is required for retraction. To this end, P-element excision was used to generate a series of chromosomal deletions that removed the gene encoding Sap18 (sap18), and the consequences on both germline and embryonic development were examined. It was found that sap18 is required maternally and zygotically, and that embryos from bcd sap18 double mutant mothers fail to undergo normal retraction of the gap gene hb at the anterior tip of the embryo. Failure to down-regulate hb leads to loss of expression of the labral determinant cap 'n' collar, resulting in severe head defects. This phenotype is further enhanced by reducing the dosage of rpd3, indicating that repression by histone deacetylases is likely to be involved. Thus, Bicoid's activity is spatially regulated in the embryo, not only by its concentration gradient along the A-P axis, but also by its interaction with a modifier (co-repressor) protein that alters its activity. The results also reveal roles for Sap18 in oogenesis, and in larval and pupal development, that are independent of Bicoid (Singh, 2005).

Zygotic sap18 mutants arrested primarily as pupae that fail to eclose. Some larval lethality was also observed, but this occurred during the third instar larval stage. Therefore, it is likely that maternally derived Sap18 is sufficient for completion of embryogenesis, and that zygotic sap18 is not involved in bcd function. The pleiotropy observed in larval and pupal phenotypes is not surprising, given that Sap18 is a member of a general co-repressor complex that probably functions during multiple stages of development. The lethality of zygotic sap18 mutants is also conditional; whereas adults were readily recovered at 22°C, none were recovered at 25°C. Although the exact cause of this temperature sensitivity is not clear, it might be due to varying strengths of protein-protein interactions involving components of the Sin3-Rpd3 complex at 22°C and at 25°C (Singh, 2005).

Two major defects were observed in maternal sap18 mutants. (1) The vast majority of eggs laid by sap18 homozygous mothers did not initiate development, and appeared unfertilized. Using sap18R7−18 germline clones, this unfertilized egg phenotype was rescued so that most embryos initiated development normally. This indicates that sap18 is required in somatic cells during oogenesis, perhaps for proper formation of the vitelline membrane or chorion, defects that would prevent fertilization. (2) Maternal sap18 mutants displayed a variety of segmentation defects, including large holes in the anterior and thoracic cuticular pattern, head abnormalities, and deleted abdominal segments. The holes in the cuticles were similar to those seen in terminal system mutants and cell death mutants, while the head defects were reminiscent of phenotypes caused by some weak bcd alleles. The inability of zygotic sap18 to rescue these defects indicates that maternal sap18 is important for embryonic development. The variety of phenotypes observed suggests multiple roles for sap18 during embryogenesis (Singh, 2005).

Despite its importance, sap18 does not seem to be absolutely essential, as evidenced by the incomplete penetrance and variable expressivity of the sap18 mutant phenotypes. In addition, it was surprising that the expression patterns of most of the segmentation genes that were examined (>15) were not detectably altered in sap18 mutant embryos. Perhaps, this is because recruitment of the Sin3-Rpd3 to promoters also occurs via other proteins in the complex, or because other unrelated co-repressors such as Groucho or dCtBP play redundant roles (Singh, 2005).

In evolutionary terms, Bicoid is a relative newcomer to the network of regulatory proteins that pattern the insect head. In Drosophila, Bicoid apparently usurped some functions carried out by the Hunchback protein in more primitive insects. Bicoid has also evolved the ability to carry out multiple distinct functions. For example, in addition to its well-studied role in regulating RNA pol II transcription, it also represses caudal mRNA translation. To carry out these diverse functions and to limit its activity in different parts of the embryo, Bicoid is likely to interact with a discrete set of modifier proteins. Included in this set are the Bin3 protein methyltransferase, whose function is still unknown, eIF4E, and Sap18 (Singh, 2005).

Although Sap18 has been found in other complexes, several findings suggest that to modulate Bicoid activity, it recruits the Sin3-Rpd3 HDAC complex: (1) Sap18 downregulates Bicoid-dependent transcription in Drosophila S2 cells, and this inhibition is reduced by the addition of trichostatin A, a known HDAC inhibitor; (2) in the present study, hb retraction was impaired in bcd sap18 double heterozygotes, resulting in de-repression of hb mRNA levels at the anterior tip of the embryo; (3) mutation of rpd3 enhanced the bcd sap18 mutant phenotypes, resulting in greater loss of hb retraction and more severe head defects (Singh, 2005).

These results lead to the following model. At the anterior of the blastoderm embryo, just prior to cellularization, Bicoid interacts with maternal Sap18 thereby recruiting the Sin3-Rpd3 HDAC complex to repress hb transcription. Repression of hb allows the anterior cap expression of the labral determinant cnc, which is required for pharyngeal development. In this model, Bicoid-Sap18 interaction must be restricted to labral and perhaps acronal regions, despite the fact that maternal sap18 mRNA is present uniformly throughout the embryo (Zhu, 2001). This spatial restriction would ensure that zygotic hb can still be activated further down the Bicoid gradient. One possibility is that the Bicoid-Sap18 interaction is inherently weak, so that it only occurs at the anterior tip of the embryo where Bicoid concentrations are the highest. Alternatively, Bicoid-Sap18 interaction might be stimulated by the terminal system tor RTK pathway, whose activity is restricted to the poles of the embryo. Thus, the role of the terminal system might be to enhance co-repressor activity of a Bicoid-Sap18 HDAC complex, but this remains unproven (Singh, 2005).

Consistent with this model, bcd sap18 and bcd sap18 rpd3 mutant embryos phenocopied tor loss-of-function mutants. In tor mutants, the labrum is missing, the dorsal bridge is not formed, and there is a collapse of the head skeleton. This may be because hb retraction by Bicoid-Sap18 repression is tor-dependent. Or, the terminal system may act in a parallel and partially redundant manner with Bicoid (see below) (Singh, 2005).

The bcd sap18 and bcd sap18 rpd3 mutants also phenocopy an unusual bcd allele, bcdE5, which has a non-sense mutation at residue 264. bcdE5 mutant embryos have a normal thorax and posterior head, but they have defects in the anterior head. The phenotype of this bcdE5 allele has been dubbed 'dispersed deletion profile' and is contrary to the phenotypes of the normal bcd allelic series where weak alleles affect the thorax, intermediate alleles affect the thorax and posterior head, and strong alleles affect the thorax and both the posterior and anterior head. In bcdE5 embryos, Bicoid activity is only affected within a sub-region of the overall fate map that is under Bicoid control, a region similar to that controlled by Sap18 in the model presented in this study. It is possible that bcdE5 encodes a protein unable to interact with Sap18, and that bcdE5 would be indifferent to the removal of maternal sap18 (Singh, 2005).

Several observations suggest that the retraction of Bicoid-dependent gene expression is likely to be more complex than indicated by this model. (1) Retraction of otd and ems expression was not reduced in bcd, sap18, rpd3 mutant combinations, and thus does not seem to involve Bicoid–Sap18 repression. Therefore, conversion of Bicoid from an activator to a repressor by Sap18 appears to be promoter-dependent. For example, the spacing of Bicoid binding sites, which is known to affect Bicoid activity, or the presence of other co-regulators on the otd and ems promoters, might prevent repression by Bicoid–Sap18. This type of differential repression has been noted for another maternal determinant, Dorsal, in its interactions with the Groucho co-repressor. Also, otd retraction depends on hkb, a target of the terminal polarity system, whereas hb retraction does not. No changes were detected in hkb expression in sap18 mutants, consistent with the failure to see effects on otd retraction. Thus, retraction of hb and otd (and ems) may occur through independent pathways (Singh, 2005).

(2) Not all maternal sap18 mutant embryos showed head defects. Even in sensitized backgrounds, the sap18 mutant head phenotypes were incompletely penetrant and temperature-sensitive, showing observable phenotypes only at 29°C. One possibility is that Bicoid also interacts with other components of the HDAC complex such as Sin3, Sap30, or the catalytic subunit Rpd3, so that elimination of one bridge protein such as Sap18 would only reduce, but not abolish repression. Or, as suggested previously, there may be an inherent redundancy of co-repressor action such that under certain conditions, that is, reduced dosages of sap18 and rpd3, other co-repressors such as Groucho or dCtBP would substitute to repress hb transcription. Finally, it is possible that part of the repressive effects of Bicoid on hb during retraction are due to a self-inhibitory domain located outside the region that interacts with Sap18 (Singh, 2005).

In summary, these data show that Sap18 is required for Bicoid-dependent retraction of hb expression in the anterior head primordium, and that retraction is likely to be the result of recruitment of a histone deacetylase complex to the hb promoter. Retraction also requires the action of the terminal polarity system, but the mechanism by which this occurs remains obscure. The simplest explanation, that Bicoid activity is downregulated as a result of phosphorylation by the tor terminal system kinases, has been ruled out. An attractive alternative is suggested by results of the present study; that the tor kinase pathway regulates either the interaction of the Sap18-Sin3/Rpd3 HDAC complex with Bicoid, or its repression activity. Future experiments will examine these possibilities, and may help explain how the terminal and anterior polarity systems converge to specify head development (Singh, 2005).

dSAP18 and dHDAC1 contribute to the functional regulation of the Drosophila Fab-7 element

The Drosophila GAGA factor [Trithorax-like (Trl)] interacts with dSAP18, which, in mammals, is a component of the Sin3-HDAC co-repressor complex. GAGA-dSAP18 interaction has been proposed to contribute to the functional regulation of the bithorax complex (BX-C). Mutant alleles of Trl, dsap18 and drpd3/hdac1 enhance A6-to-A5 transformation indicating a contribution to the regulation of Abd-B expression at A6. In A6, expression of Abd-B is driven by the iab-6 enhancer, which is insulated from iab-7 by the Fab-7 element. GAGA, dSAP18 and dRPD3/HDAC1 co-localize to ectopic Fab-7 sites in polytene chromosomes, and mutant Trl, dsap18 and drpd3/hdac1 alleles affect Fab-7-dependent silencing. Consistent with these findings, chromatin immunoprecipitation analysis shows that, in Drosophila embryos, the endogenous Fab-7 element is hypoacetylated at histones H3 and H4. These results indicate a contribution of GAGA, dSAP18 and dRPD3/HDAC1 to the regulation of Fab-7 function (Canudas, 2005).

The conclusion that GAGA, dSAP18 and dRPD3/HDAC1 contribute to the function of the Fab-7 element of BX-C is based on the following observations:

  1. the localization of GAGA, dSAP18 and dRPD3/HDAC1 at ectopic Fab-7 elements (Canudas, 2005).
  2. the effects of Trl, dsap18 and drpd3/hdac1 mutations on Fab-7-dependent silencing. Ectopic Fab-7 constructs are known to mediate silencing of flanking reporter genes both in cis, as in heterozygous GCD6 flies, as well as in trans, as in 5F24 flies, where silencing is pairing-sensitive being observed only when the transgene is in a homozygous state. This study shows that Trl, dsap18 and drpd3/hdac1 mutations affect both cis- and trans-silencing mediated by Fab-7 (Canudas, 2005).
  3. the homeotic A6-to-A5 transformation observed in flies heterozygous for various Trl, dsap18 and drpd3/hdac1 mutant alleles and hemizygous for Df(3R)sbd45, which uncovers dsap18. This homeotic transformation results from the ectopic repression of the iab-6 enhancer at A6 that is insulated from the repressed iab-7 enhancer by the Fab-7 element. The fact that this homeotic transformation is very infrequent in hemizygous Df(3R)sbd45 flies, as well as in the heterozygous mutants, demonstrates that it is directly associated to the Trl, dsap18 and drpd3/hdac1 mutations. Moreover, a single copy of a transgene expressing dsap18 significantly rescues this phenotype. The results also indicate that an unidentified element(s) contained within Df(3R)sbd45 is also contributing to the establishment of the phenotype. In addition to sap18, Df(3R)sbd45 uncovers at least 11 other genes including the trithorax gene, taranis. However, the homeotic transformation described in this study does not appear to be associated to a loss of taranis function since no transformation is observed in flies trans-heterozygous for a null taranis allele and Trl, dsap18 or drpd3/hdac1 mutations (Canudas, 2005).

Together, these results indicate a contribution of GAGA, dSAP18 and dRPD3/HDAC1 to the structural and functional properties of Fab-7. What could this contribution be? Several models might account for these results. Fab-7 is known to contain two functional elements: a PRE, which is required for Pc-dependent silencing, and an adjacent boundary element that insulates iab-6 from iab-7. The finding that, in heterozygous GCD6 flies, mutant Trl, dsap18 and drpd3/hdac1 alleles enhance cis-silencing imposed by Fab-7 suggests that their functions might antagonize Pc-dependent silencing. Several observations, however, make this hypothesis unlikely: (1) at some PREs, GAGA helps recruitment of PcG complexes and contributes to silencing; (2) dRPD3/HDAC1 was shown to be a component of several PcG complexes, and genetic analysis indicates a contribution to homeotic silencing; (3) in mammals, SAP18 acts as a repressor when targeted to an active promoter (Canudas, 2005).

An alternative possibility is that GAGA, dSAP18 and dRPD3/HDAC1 contribute to the function of the Fab-7 boundary element. In fact, the Fab-7 boundary contains several GAGA-binding sites that are required for its enhancer blocking activity and, it is hypoacetylated at histones H3 and H4. In GCD-6 flies, the Fab-7 boundary element is located proximal to the reporter mini-white gene with respect to the PRE so that it might help to insulate the reporter gene from repression by the PRE. In this context, mutations that affect boundary function would result in a less efficient insulation and, therefore, would enhance silencing (Canudas, 2005).

In contrast to the enhancer effect observed in heterozygous GCD6 flies, mutations in Trl, dsap18 and drpd3/hdac1 suppress pairing-dependent trans-silencing in transgenic 5F24(25,2) flies. A contribution to boundary-functions might also account for this effect. Pairing-sensitive trans-silencing results from long-distance chromosomal interactions that involve the association of the transgenes with each other and with the endogenous Fab-7 element, even when located in different chromosomes. These long-distance interactions that require the contribution of PcG proteins might be facilitated by a functional boundary element as has been described for the gypsy insulator (Canudas, 2005).

The incomplete A6-to-A5 homeotic transformation observed in the presence of Trl, dsap18 and drpd3/hdac1 mutations might also reflect a contribution to the boundary function of Fab-7 as, in the mutant conditions, it might not properly insulate the iab-6 enhancer from the repressing activity of the Fab-7 PRE, thereby becoming partially inactivated. Interestingly, mutations that delete the Fab-7 boundary but not the PRE produce, in addition to strong A6-to-A7 transformation, incomplete A6-to-A5 transformation. Moreover, replacement of the Fab-7 boundary by the gypsy or the scs insulator (both of which are not functional in the context of BX-C) results in complete A6-to-A5 transformation (Canudas, 2005).

The results indicate that GAGA, dSAP18 and dRPD3/HDAC1 have similar effects on the functional properties of Fab-7 suggesting a functional link. A physical interaction between GAGA and dSAP18 has been reported. Moreover, in mammals, SAP18 is associated with the Sin3-HDAC co-repressor complex and, in Drosophila, dSAP18 modulates bicoid activity through the recruitment of dRPD3/HDAC1 and it is required to suppress bicoid activity in the anterior tip of the embryo. In this context, it is tempting to speculate that GAGA helps in the recruitment of dSAP18 and dRPD3/HDAC1 to Fab-7 resulting in a concerted contribution to its boundary function (Canudas, 2005).

In mammals, SAP18 is also associated with ASAP, a protein complex involved in RNA processing. In Drosophila, dSAP18 may also participate in RNA processing; in cultured S2 cells, a large proportion of dSAP18 co-immunoprecipitates with factors that participate in RNA processing. It is possible that, in response to cellular signals, the association of dSAP18 to different protein complexes would be regulated during development and/or cell cycle progression (Canudas, 2005).

Trithorax-like interaction with SAP18, a Sin3-associated polypeptide

Drosophila SAP18 (accepted FlyBase name: Bicoid interacting protein 1), a polypeptide associated with the Sin3-HDAC co-repressor complex, has been identified in a yeast two-hybrid screen as capable of interacting with the Drosophila GAGA factor. The interaction was confirmed in vitro by glutathione S-transferase pull-down assays using recombinant proteins and crude SL2 nuclear extracts. The first 245 residues of GAGA, including the POZ domain, are necessary and sufficient to bind dSAP18. In polytene chromosomes, Drosophila SAP18 and GAGA co-localize at a few discrete sites and, in particular, at the bithorax complex where GAGA binds some silenced polycomb response elements. When the Drosophila SAP18 dose is reduced, flies heterozygous for the GAGA mutation Trl67 show the homeotic transformation of segment A6 into A5, indicating that GAGA-dSAP18 interaction contributes to the functional regulation of the iab-6 element of the bithorax complex. These results suggest that, through recruitment of the Sin3-HDAC complex, GAGA might contribute to the regulation of homeotic gene expression (Espinas, 2000).

The identity of dSAP18 with either human (hSAP18) or C. elegans (cSAP18) SAP18 is high, ~60% and 47%, respectively. The three polypeptides show high homology throughout their sequences, except for the most N- (1-15) and C-terminal (138-150) residues, and a central region (residues 32-45). Two specific regions, RI (16-31) and RII (65-89), show a very high degree of conservation with a similarity >80%. A third region, RIII (123-137), also shows significant similarity (80%), but in this case the identity is lower (47%) than for regions RI (81%) and RII (67%) (Espinas, 2000).

GAGA is organized into several functionally distinct domains. A single zinc finger is involved in nucleic acid recognition. In addition to this central DNA binding domain (DBD), GAGA carries a C-terminal glutamine-rich domain (Q-domain), which is involved in transcription activation, and a highly conserved N-terminal POZ domain, which mediates protein-protein interactions. A relatively long (140 amino acids) region of unknown function(s) links the POZ and DBD domains. Little is known about the interaction of GAGA with other nuclear proteins. The POZ domain of GAGA has been shown to support homomeric as well as heteromeric interactions with other POZ-containing proteins, such as tramtrack (ttk). The first 245 residues of GAGA are necessary and sufficient for binding dSAP18, and efficient GAGA-dSAP18 interaction requires the contribution of both the POZ and linking domains of GAGA. Not all regions of dSAP18 contribute equally to its interaction with GAGA, and residues 73-113, which include most of the highly conserved RII region, are mainly responsible for binding to GAGA (Espinas, 2000).

SAP18 was identified as a polypeptide associated with the mammalian transcriptional repressor Sin3. The core mSin3 complex contains a total of seven polypeptides, which include the histone deacetylases HDAC1 and HDAC2, RbAp48 and RbAp46, and SAP30 and SAP18. Recruitment of the Sin3-HDAC complex to specific target genes appears to rely on its interaction with sequence-specific DNA binding proteins, since none of the known components of the complex are capable of binding DNA. SAP30 and SAP18 could mediate some of these interactions. mSAP30 binds both mSin3 and N-CoR, and is required for N-CoR-mediated repression by a set of sequence-specific DNA-binding transcription factors. SAP18 could also be involved in interactions with sequence-specific DNA binding proteins. It is known that SAP18 interacts directly with mSin3. Since POZ is a highly conserved structural domain, it is likely that similar interactions would be observed with other POZ domains. Actually, several sequence-specific transcriptional repressors carry POZ domains, some of which are also found to interact with N-CoR and SMRT. Most likely, formation of a stable complex requires multiple interactions between its various components (Espinas, 2000 and references therein).

Contrary to most POZ-containing proteins, the Drosophila GAGA factor acts as a transcriptional activator. Its interaction with a component of the Sin3 co-repressor complex indicates that GAGA might also act as a repressor in some cases. In this respect, the presence of GAGA at some silenced PREs of the bithorax and antennapedia complexes might be especially revealing. Interestingly, though the immunostaining patterns of GAGA and dSAP18 show only a limited general overlapping in polytene chromosomes, the two proteins co-localize at the region of the bithorax complex (BX-C), suggesting a possible contribution of GAGA-dSAP18 interaction to BX-C regulation. Consistent with this possibility, a genetic interaction between Trl and a deficiency that uncovers dSAP18 has been observed. Flies heterozygous for the Trl67 mutation and hemizygous for Df(3R)sbd26 show a homeotic transformation of the sixth abdominal segment into the fifth as indicated by the presence in the sixth sternite of several bristles in the vast majority of the individuals. Results presented indicate that GAGA-dSAP18 interaction has a significant contribution to the functional regulation of the iab-6 element of BX-C (Espinas, 2000).

The concurrent presence of GAGA and Polycomb at some silenced PREs is surprising since, as derived from genetic analysis, these two proteins are expected to have opposing functions on the regulation of the expression of the homeotic genes. Acting at the level of the core promoter elements, GAGA is likely to activate transcription of the homeotic genes. However, functional trithorax response elements (TREs) are frequently found in the vicinity of PREs and a contribution of GAGA to the functional regulation of several segment-specific cis-regulatory regions of the bithorax complex has been reported (Espinas, 2000 and references therein).

It is still uncertain whether GAGA helps to establish the repressed or the active state of these elements. GAGA has been shown to contribute to the relief of repression at the Fab-7 element, and the homeotic transformations described here and elsewhere are also consistent with a role in activation. However, in the case of the iab-7 and bxd PREs, GAGA has been shown to contribute to silencing and the genetic interactions observed between some Pc and Trl alleles also suggest a contribution to repression. The results presented here indicate that GAGA might participate in the recruitment of the Sin3-HDAC co-repressor complex to some PREs, but that contrary to what would be anticipated for such an interaction, it contributes to the relief of repression at the iab-6 element. The same phenotype is observed in flies homozygous for the hypomorph Trl13C allele. Interestingly, some rpd3 alleles behave as enhancers of PEV, also leading to an increase in repression. It is possible that by modifying chromatin structure, GAGA-SAP18 interaction could contribute to the establishment of the domain boundaries that insulate different cis-regulatory elements, rather than to the formation of the repressed or active states themselves (Espinas, 2000).

Drosophila SAP18, a member of the Sin3/Rpd3 histone deacetylase complex, interacts with Bicoid and inhibits its activity

Bicoid directs anterior development in Drosophila embryos by activating different genes along the anterior-posterior axis. However, its activity is down-regulated at the anterior tip of the embryo, in a process known as retraction. Retraction is under the control of the terminal polarity system, and results in localized repression of Bicoid target genes. A Drosophila homolog of human SAP18 (Sin3A-associated polypeptide p18), a member of the Sin3A/Rpd3 histone deacetylase complex (HDAC), is described. Termed Bicoid interacting protein 1 (Bip1), the SAP18 homolog interacts with Bicoid in yeast and in vitro, and is expressed early in development coincident with Bicoid. In tissue culture cells, Bip1 inhibits the ability of Bicoid to activate reporter genes. These results suggest a model in which Bip1 interacts with Bicoid to silence expression of Bicoid target genes in the anterior tip of the embryo (Zhu, 2001).

A cDNA encoding Bin1 was identified using a custom two-hybrid selection in which Bicoid was bound to DNA via its homeodomain. The 5' end of the bin1 cDNA was cloned by RACE and a full-length cDNA sequence was assembled. The bin1 cDNA encodes a 150-amino-acid protein with a predicted molecular weight of 17.3 kDa. The protein is 58% identical to the human and murine SAP18 proteins and 42% identical to a C. elegans ORF. The Drosophila genome sequence does not predict any other homologs. A search of the Berkeley Drosophila Genome Project database revealed an EP insertion line EP(3)3462 in which an EP-transposon is inserted 259 bp upstream of the bin1 start codon, and 151 bp upstream of the transcription initiation site. This insertion is within the 5' UTR of nebula, an ORF oriented opposite to that of bin1 (Zhu, 2001).

LexA-Bicoid fusion proteins were used to map the regions within Bicoid that are important for the Bin1-Bicoid interaction. In these experiments, Bin1 was fused to the B42 activation domain. Results from these assays show that interaction with Bin1 does not require the Bicoid acidic activation domain (AD), or the polyglutamine (Q) or polyalanine (A) domains. The homeodomain is not sufficient for interaction, but seems to be required along with flanking regions, each of which contributes modestly to the interaction. Thus, the interaction requires two distinct regions of Bicoid, aa 1-95 and aa 163-246. To test whether Bin1 interacts directly with Bicoid in vitro, the full-length Bin1 was expressed as a GST fusion protein in Escherichia coli. GST-Bin1 was attached to glutathione beads and used in pull-down experiments with 35 S-methionine-labeled, full-length Bicoid generated by in vitro translation. GST-Bin1 interacts with Bicoid in this system. Thus, Bin1 interacts directly with Bicoid in vitro (Zhu, 2001).

If Bin1 is required for Bicoid function, then its protein expression pattern should overlap with that of Bicoid temporally and spatially. Bicoid is translated from maternally deposited mRNA shortly after egg laying. After 3 h of development, the protein level begins to diminish, and after 4 h, Bicoid is undetectable. To determine when the Bin1 gene is expressed, Northern analysis was carried out using mRNA isolated from unfertilized eggs and from 0- to 2-h, 2- to 4-h, and 4- to 24-h developing embryos. A Bin1 mRNA of about 500 nt is detected in unfertilized eggs and in early embryos. The mRNA levels peak around the cellularization to early gastrulation stages (2-4 h). These results indicate that Bin1 is transcribed both maternally and zygotically, and the Bin1 mRNA is present at the time that Bicoid is present (Zhu, 2001).

To determine the spatial distribution of Bin1 mRNA within the early embryo, whole-mount in situ hybridization was carried out using anti-sense. In contrast to the highly localized BCD mRNA, Bin1 mRNAs are distributed throughout the early embryo and in the unfertilized egg. Thus, the expression pattern of Bin1 overlaps spatially with that of Bicoid protein, which is detectable over the anterior two-thirds of the embryo. The temporal and spatial expression pattern of Bin1 mRNA suggests that Bin1 protein is present throughout the embryo, although proof of protein localization will require anti-Bin1 immunostaining (Zhu, 2001).

Based on the role of human SAP18 in transcription repression by HDAC complexes, tests were performed to see whether over-expression of Bin1 inhibits Bicoid-dependent transcription in Drosophila S2 cells. In this assay, plasmids expressing Bicoid and Bin1 were co-transfected along with a Bicoid binding site-CAT reporter construct. The results indicate a dose-sensitive inhibition of Bicoid-dependent transcription by Bin1. The effect is greater at lower Bicoid concentrations, suggesting that the ratio of Bin1 to Bicoid is important for the effect (Zhu, 2001).

The Sin3A Rpd3 histone deacetylase complex is conserved in Drosophila. Both Sin3 and an Rpd3 homolog (HDAC1) have been identified in Drosophila and are required for embryogenesis. By analogy with mammalian systems, Bin1 is likely to function in co-repression as part of a Drosophila Sin3/HDAC1 complex. It is proposed that interaction with Bin1 recruits the HDAC complex to DNA, converting Bicoid from an activator into a repressor, or at least neutralizing its ability to stimulate expression of its target genes. In this model, interaction of Bicoid with Bin1 would be stimulated by the action of the terminal polarity-system kinases. For example, phosphorylation of either Bicoid or Bin1 might trigger a conformational change that strengthens their interaction. The Bin1-Bicoid complex would then recruit Sin3/HDAC1 to down-regulate Bicoid's transcription activity beginning at late cellularization stages. In this way, Bicoid-dependent gene expression could be down-regulated exclusively at the anterior tip of the embryo, where the Bicoid concentration is high and the terminal system is active, resulting in the observed retraction (Zhu, 2001).

Human SAP18 has been found to interact with a cAMP-GEF protein. cAMP-GEF proteins function in MAPK signal transduction pathways to activate the GTPases Rap1 and Ras, which in turn leads to activation of Raf kinases (MAPKKK). Members of this pathway are present in Drosophila, including two putative proteins similar to human cAMP-GEFs, CG3427, located at 42C4-5, and CG9494, located at 26C3, as well as dRap1 (Roughened) and Raf kinase (Pole hole protein), which is the kinase downstream of the Torso receptor in the terminal system. By analogy with human SAP18, Drosophila Bin1 might interact with a cAMP-GEF, and thereby be linked directly to the terminal system MAP-kinase pathway. For example, interaction of Bin1 with cAMP-GEF might result in phosphorylation of Bin1 by Raf upon stimulation of the Torso receptor tyrosine kinase. This, in turn, might stimulate Bin1 to interact with Bicoid and trigger recruitment of the HDAC complex to Bicoid-regulated promoters (Zhu, 2001).

Bin1 has also been identified as a protein that interacts with Enhancer of Zeste, E(z), (L. Ding and R. Jones, personal communication to Zhu, 2001), a Polycomb group protein important for maintenance of repression of homeotic genes, and with GAGA factor, the trithorax-like gene product required for activation of homeotic genes. These and other examples suggest that control of expression of homeobox genes by histone deacetylases is important for embryogenesis. Histone deacetylases may also alter homeodomain protein activity by direct interaction. Mobilization of the EP-transposon insertion near Bin1 should make it possible to generate mutant alleles, which will be important for studying the role of Bin1 in development (Zhu, 2001).

Drosophila Enhancer of zeste protein interacts with dSAP18

The Drosophila Enhancer of zeste [E(z)] gene encodes a member of the Polycomb group of transcriptional repressors. This study provides evidence for direct physical interaction between E(Z) and dSAP18, which previously has been shown to interact with Drosophila GAGA factor and Bicoid proteins. dSAP18 shares extensive sequence similarity with a human polypeptide originally identified as a subunit of the SIN3A-HDAC (switch-independent 3-histone deacetylase) co-repressor complex. Yeast two-hybrid and in vitro binding assays demonstrate direct E(Z)-dSAP18 interaction and show that dSAP18 is capable of interacting with itself. Co-immunoprecipitation experiments provide evidence for in vivo association of E(Z) and dSAP18. Gel filtration analysis of embryo nuclear extracts shows that dSAP18 is present in native protein complexes ranging from approximately 1100 to approximately 450 kDa in molecular mass. These studies provide support for a model in which dSAP18 contributes to the activities of multiple protein complexes, and potentially may mediate interactions between distinct proteins and/or protein complexes (Wang, 2002).

Suppressor of Fused represses Gli-mediated transcription by recruiting the SAP18-mSin3 corepressor complex

The Suppressor of Fused [Su(fu)] protein plays a conserved role in the regulation of Gli transcription factors of the hedgehog (Hh) signaling pathway that controls cell fate and tissue patterning during development. In both Drosophila and mammals, Su(fu) represses Gli-mediated transcription, but the mode of its action is not completely understood. Recent evidence suggests that Su(fu) physically interacts with the Gli proteins and, when overexpressed, sequesters Gli in the cytoplasm. However, Su(fu) also traverses into the nucleus under the influence of a serine-threonine kinase, Fused (Fu), and has the ability to form a DNA-binding complex with Gli, suggesting that it has a nuclear function. This study reports that the mouse homolog of Su(fu) [mSu(fu)] specifically interacts with SAP18, a component of the mSin3 and histone deacetylase complex. In addition, mSu(fu) functionally cooperates with SAP18 to repress transcription by recruiting the SAP18-mSin3 complex to promoters containing the Gli-binding element. These results provide biochemical evidence that Su(fu) directly participates in modulating the transcriptional activity of Gli (Cheng, 2002).

Drosophila dSAP18 is a nuclear protein that associates with chromosomes and the nuclear matrix, and interacts with pinin, a protein factor involved in RNA splicing

SAP18 is a highly conserved protein that was proposed to be involved in multiple cellular processes from autophagy to gene regulation and mRNA processing. In Drosophila, dSAP18 is a predominantly nuclear protein that associates to both chromosomes and the nuclear matrix. dSAP18 becomes nuclear early during development, at the onset of cellularization, and remains so all through embryo development. dSAP18 is also nuclear in salivary glands, ovaries and cultured S2 cells. dSAP18 forms a complex with the Drosophila homolog of pinin (Drosophila Pinin), a protein factor involved in mRNA splicing. dSAP18-dPnn interaction was confirmed in vivo, through co-immunoprecipitation experiments, as well as in vitro, through GST pull-down assays. These results are discussed in the context of the possible functions played by SAP18 (Costa, 2006).

SAP18 promotes Krüppel-dependent transcriptional repression by enhancer-specific histone deacetylation

Body pattern formation during early embryogenesis of Drosophila relies on a zygotic cascade of spatially restricted transcription factor activities. The gap gene Krüppel ranks at the top level of this cascade. It encodes a C2H2 zinc finger protein that interacts directly with cis-acting stripe enhancer elements of pair rule genes, such as even skipped and hairy, at the next level of the gene hierarchy. Krüppel mediates their transcriptional repression by direct association with the corepressor Drosophila C terminus-binding protein (dCtBP). However, for some Krüppel target genes, deletion of the dCtBP-binding sites does not abolish repression, implying a dCtBP-independent mode of repression. This study identified Krüppel-binding proteins by mass spectrometry and found that SAP18 can both associate with Krüppel and support Krüppel-dependent repression. Genetic interaction studies combined with pharmacological and biochemical approaches suggest a site-specific mechanism of Krüppel-dependent gene silencing. The results suggest that Krüppel tethers the SAP18 bound histone deacetylase complex 1 at distinct enhancer elements, which causes repression via histone H3 deacetylation (Matyash, 2009).

This study provides evidence that Kr exerts transcriptional repression not only by association with the corepressor dCtBP but also by site-specific deacetylation of histones, a mechanism that involves an interaction between Kr and dSAP18. The dual mode of Kr-dependent repression might explain earlier studies showing that Kr represses eve stripe 2 expression, but not h stripe 7 expression, in a dCtBP-dependent manner. Consistent with these observations, a mutant Kr protein that lacks dCtBP-binding sites still associates with dSAP18, which in turn interacts with the Sin3A-HDAC1 repressor complex. dSAP18 was also shown to bind the homeodomain transcription factor Bicoid, causing repression of anterior gap genes such as hunchback in the late Drosophila blastoderm embryo. SAP18-dependent repression involves histone deacetylase both in flies and mammals, and SAP18 that links the HDAC1 complex with sequence-specific transcriptional repressors bound to chromatin is also found in plants. These results are consistent with such a SAP18-dependent mode of Kr-dependent repression that provides target gene-specific repression. Because both dCtBP and SAP18 are uniformly distributed in the embryo, it will be important to learn how the eve stripe 2 and the h stripe 7 enhancer distinguish between the dCtBP- or SAP18-dependent modes of repression. One possibility is that differential packing of the enhancer DNA into nucleosomes might account for the difference in susceptibility to the SAP18/HDAC1-mediated repression (Matyash, 2009).

dSAP18 binds to three distinct regions of Kr, including the 42-amino acid-long repressor region, which is conserved in Kr homologs of all Drosophila species. However, as observed for dCtBP, dSAP18 alone cannot account for Kr-dependent repression of h7-lacZ, because prolonged expression of Kr is able to overcome the lack of dSAP18 activity as observed for the h7 element in dSAP18 mutants. Therefore, it is likely that the full spectrum of Kr-dependent repression is mediated redundantly, employing at least two different corepressors that involve different modes of repression (Matyash, 2009). In vitro, dSAP18 binds to the sequence motif 344RRRHHL349 of Kr and to a similar motif (143RRRRHKI149) of Bicoid; the latter is consistent with the results reported by Zhu (2001). In both proteins, the dSAP18-binding sites are localized in the C-terminal portion of their DNA-binding domains. Thus, when acting from weak binding sites in vivo, transcription factors might be able to form strong complexes with dSAP18. In fact, Bicoid-dependent repression of hunchback, which depends on both SAP18 and HDAC1 (Singh, 2005), occurs only at the very anterior tip of blastoderm embryos where the Bicoid concentration is highest and the target gene enhancers contain multiple weak Bicoid-binding sites (Matyash, 2009).

dSAP18 also interacts with the histone-specific H3K27 methyl-transferase E(z) (Enhancer of zeste) (Wang, 2002), a component of the polycomb group protein complex, and with the GAGA factor, a transcription factor of the trxG (trithorax group) protein complex. Thus, dSAP18 is capable of interacting with two regulatory protein complexes that have antagonistic functions in gene regulation. Whereas the polycomb group complex acts as a repressor of homeotic genes in ectopic locations, the trxG complex is required for activation and maintenance of their transcription. However, this clear-cut distinction between polycomb group and trxG functions has been questioned, because polycomb group and trxG group members were shown to act both as context-dependent repressors and activators of transcription, and factors with such dual functions include both the E(z) and GAGA factor proteins. In fact, interactions between dSAP18 and GAGA factor at the iab-6 element of the bithorax complex, for example, were shown to cause transcriptional activation and not repression (Matyash, 2009).

This study suggests that Kr mediates repression through at least two pathways involving either dCtBP or SAP18. dCtBP-dependent and -independent repression of the transcription factors Knirps and Hairless exert quantitative effects, whereas Kr distinguishes dCtBP and dSAP18 recruitment at different enhancers. It was observed, however, that the loss of SAP18 activity does not affect the pattern of eve stripe expression and that prolonged Kr can suppress h7-lacZ expression in the absence of dSAP18. Thus, although both dSAP18 and dCtBP act independently from each other, the two corepressors, or other yet unknown corepressors, can functionally substitute for each other under forced conditions. However, their mode of repression appears to involve different mechanisms. One mechanism is exemplified by the dCtBP-dependent repression of eve-stripe 2 and not yet established at the molecular level. dCtBP-dependent repression does not act via unleashing local heterochromatization, does not require dHDAC1 activity, and is insensitive to the HDAC inhibitor TSA. Consistently, coimmunoprecipitation studies failed to detect HDAC activity in the dCtBP immunoprecipitates, histone H3 remained acetylated in dCtBP-deficient embryos, and transcription was not repressed. Other studies, however, implied an association of dCtBP with HDACs. Thus, the mechanism of the dCtBP mode of repression is not yet fully understood (Matyash, 2009).

The results of this study showing a lack of H3 deacetylation at the eve stripe 2 enhancer in response to Kr repression are consistent with the argument that eve stripe 2-mediated repression involves the corepressor CtBP. The second, dCtBP-independent mode of Kr-dependent repression, as exemplified by the h stripe 7 element (and possibly also eve stripes 1, 3, and 4) does require both dSAP18 and HDAC1 activities. In support of this mode of repression, the following phenomena were observed in Kr-overexpressing embryos (1) a dSAP18-dependent loss of K9,14H3 acetylation on the h stripe 7 element, (2) an increased resistance of the h7 enhancer DNA to sonication, and (3) SAP18-dependent repression of the h7 reporter gene in response to Kr activity. These Kr-dependent effects were dependent on HDAC1 enzymatic activity as revealed by experiments using the HDAC1 inhibitor, TSA. These results therefore suggest that dSAP18-dependent repression by Kr involves structural changes of chromatin, such as compaction or condensation, likely to be caused by site-specific heterochromatization in response to enhancer-specific HDAC1 activity (Matyash, 2009).


REFERENCES

Search PubMed for articles about Drosophila Sap18

Canudas, S., et al. (2005). dSAP18 and dHDAC1 contribute to the functional regulation of the Drosophila Fab-7 element. Nuc. Acids Res. 33(15): 4857-64. PubMed ID: 16135462

Cheng, S. Y. and Bishop, J. M. (2002). Suppressor of Fused represses Gli-mediated transcription by recruiting the SAP18-mSin3 corepressor complex. Proc. Natl. Acad. Sci. 99: 5442-5447. PubMed ID: 11960000

Costa, E., et al. (2006). Drosophila dSAP18 is a nuclear protein that associates with chromosomes and the nuclear matrix, and interacts with pinin, a protein factor involved in RNA splicing. Chromosome Res. 14(5): 515-26. PubMed ID: 16823614

Espinas, M. L., Canudas, S., Fanti, L., Pimpinelli, S., Casanova, J. and Azorin, F. (2000). The GAGA factor of Drosophila interacts with SAP18, a Sin3-associated polypeptide. EMBO Rep. 1: 253-259. PubMed ID: 11256608

Matyash, A., et al. (2009). SAP18 promotes Krüppel-dependent transcriptional repression by enhancer-specific histone deacetylation. J. Biol. Chem. 284(5): 3012-20. PubMed ID: 19049982

Schwerk, C., et al. (2003). ASAP, a novel protein complex involved in RNA processing and apoptosis. Mol. Cell. Biol. 23: 2981-2990. PubMed ID: 12665594

Singh, N., Zhu, W. and Hanes, S. D. (2005). Sap18 is required for the maternal gene bicoid to direct anterior patterning in Drosophila melanogaster. Dev. Biol. 278(1): 242-54. PubMed ID: 15649476

Wang, L. Ding, L., Jones, C. A. and Jones, R. S. (2002). Drosophila Enhancer of zeste protein interacts with dSAP18. Gene 285: 119-125. PubMed ID: 12039038

Zhang, Y., et al. (1997). Histone deacetylases and SAP18, a novel polypeptide, are components of a human Sin3 complex. Cell 89: 357-364. PubMed ID: 9150135

Zhu, W., Foehr, M., Jaynes, J. B. and Hanes, S. D. (2001). Drosophila SAP18, a member of the Sin3/Rpd3 histone deacetylase complex, interacts with Bicoid and inhibits its activity. Dev. Genes Evol. 211: 109-117. PubMed ID: 11455422


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